The protozoan parasites of the genus Leishmania are the causative agents of visceral leishmaniasis (VL), also called kala-azar (KA). KA is a symptomatic infection of the liver, spleen and bone marrow caused by organisms of Leishmania donovani complex. The annual incidence and prevalence of cases of visceral leishmaniasis worldwide is 0.5 million and 2.5 million respectively. Of these 90% of cases occur in India, Nepal, Bangladesh and Sudan. The causative organism in the Indian subcontinent and Africa is L. d. donovani, while in the Mediterranean basin and South America it is L. d. infantum. 
PKDL (Post kala-azar dermal leishmaniasis) is an unusual dermatosis that develops as a sequel of KA, producing gross cutaneous lesions in the form of hypopigmented macules, erythema and nodules. The disease is relatively common in the Indian subcontinent and less frequent in East Africa, but exceptional in the American and European continents. Detection and characterization of Leishmania from patients of both KA and PKDL is important for deciding treatment regimens as well as for understanding the disease epidemiology.
Current diagnostic methods based on parasite detection (stained smears, culture and histopathology) and immunological methods (DAT, ELISA etc.) have several limitations including low sensitivity and specificity. Procedures for demonstration of the parasite in spleen or bone marrow in KA and in skin lesions in PKDL are invasive and often not sensitive enough. Immunological methods fail to distinguish between past and present infections and are not reliable in case of immuno-compromised patients. Furthermore, both of these methods do not address the problem of species identification, which is important to determine appropriate treatment regimens and designing control measures. Procedures involving the use of monoclonal antibodies, isoenzyme and schizodeme analysis and DNA hybridization have to be resorted to. Most of these procedures are tedious and require massive cultures of parasites. There is, therefore, an urgent need to develop diagnostic procedures that are simple, sensitive and specific.
In recent years PCR based diagnostic methods have been described for leishmaniasis, with a wide range of sensitivity and specificity. An excellent target for a sensitive and rapid detection method is the kinetoplast mini-circle DNA, which are present at thousands of copies per cell. The mini-circles have been used as targets for selective amplification of parasite DNA in various studies Aviles, H., A. Belli, R. Armijos, F. P. Monroy, and E. Harris J. Parasitol. 1999, 85:181-187; BhattacharyaR., K. Das, S. Sen, S. Roy, and H. K. Majumder. 1996. Microbiol. Lett. 135:195-200; Nuzum, E., F. White III, C. Thakur, R. Dietze, J. Wages, M. Grogi, and J. Berman. 1995 J. Lnf. Dis. 171: 751-754; Rogers M. R, Popper S. J., and Wirth D. F. 1990. Exp. Parasitol. 71: 267-275; Smyth, A. J., A. Ghosh, Md. Q. Hassan, D. Basu, M. H. L. De Bruijn, S. Adhya K. K. Mallik, andD. C. darker. 1992, Parasitol 105: 183-192.
Wirth and Pratt (Proc Natl Acad Sci USA. 79:6999-7003 (1982) have described a hybridization assay for the detection of Leishmania parasites using probes to parasite kinetoplast DNA. This assay detects parasites in cutaneous lesions at a sensitivity level of 1,000-10,000 parasites per biopsy specimen. The specimens are collected by touch-blotting of nitrocellulose sheets over a small area of infected skin. However, this method is not sensitive enough to detect small numbers of parasites and relies on probes that have to be purified from the parasites themselves. This requires growth of these organisms in large quantities in the laboratory.
A more sensitive assay has been sought for Leishmania parasites which will be sensitive, specific, and thus be useful in early diagnosis of infection, will identify the species of parasites more likely to induce severe disease, and aid evaluation of chemotherapy and screening of blood bank samples.
To this end, the Applicant has exploited the kinetoplast mini-circle DNA present in leishmania parasites and used these kinetoplast sequences to develop novel oligonucleotide primers which are extremely sensitive and capable of detecting leishmanial parasites from the peripheral blood and skin lesions of infected patients.